CD8+ T cells (TCD8+) recognize class I molecules of the major histocompatibility complex (MHC) bearing peptides of 8 to 10 residues derived from cytosolic proteins. These cells are a bulwark of host defenses to infectious agents and tumors, and it is critical to understand how antigenic peptides are generated by cells if we are to improve existing vaccines and develop new vaccines and treatments for infectious and neoplastic diseases. Very little is known about how cells produce antigenic peptides from proteins. Previous results from our laboratory indicate that the endoplasmic reticulum (ER) is capable of trimming longer peptides that are imported from the cytosol. We have used a novel strategy to characterize the proteolytic capacity of the ER and its relevance to antigen processing. Our findings suggest that the ER is capable of trimming peptides to the sizes preferred by MHC class I molecules.